Adherent mammalian cells cultured in vitro are typically removed from the culture surface by use of trypsin, a protease which cleaves molecules involved in adherence. However, it has be found that after several treatments with trypsin, the cells become non-adherent and lose some normal functions. Additionally, the addition of a protease to a cell culture may cause other cellular damage, and if possible should be avoided. However, few alternatives are available.
The invasive potential of a cancer cell is extremely important. The traditional method of looking at such invasive potential of living cells is by using in vitro assays. This requires an in vitro assay and it typically takes about 3 weeks for results to be obtained [J. Behrens, et al., J. Cell Biol., 108:2435 (1989)]. This can be an extremely long time period where rapid diagnosis is desired. Accordingly, it would be desirable if there was a rapid method for assaying cells for their invasive potential.
Control of cell shape by extracellular matrix (ECM), molecules outside the cell, is critical for regulation of cell growth [J. Folkman, et al., Nature, 273:45 (1978); D. Ingber, Proc. Natl. Acad. Sci. U.S.A., 87:3579 (1990)] differentiation [M. Bissel, et al., J. Theor. Biol., 99:31 (1982); Mooney, et al., J. Cell Physiol., 151:497 (1992)] cell polarity [D. Ingber, et al., Am. J. Path., 122:129 (1986)] and tissue pattern formation [D. Ingber, et al., Proc. Natl. Acad. Sci. U.S.A., 78:3901 (1981); D. Ingber, et al., J. Cell. Biol., 109:317 (1989)]. These functions depend on the close cooperation of the ECM, protein on the surface of the cell, for example, cell adhesion molecules such as integrins, and proteins inside the cell, including the proteins of the cytoskeleton (CSK).
CSK structure has been shown to be associated with, for example, cell motility, differentiation, mitosis and adhesion. [T. P. Stossel, Science, 260:1086 (1993); J. S. Hyams, and B. R. Brinkley, eds. "Mitosis: Molecules And Mechanisms," Academic Presss, San Diego (1989).] The function of CSK structure is tightly coupled to CSK mechanical properties. One very important parameter of CSK mechanical properties is CSK stiffness. Accordingly, the ability to regulate or control the cytoskeletal stiffness of cells, both in vivo and in vitro, is important not only to advance understanding of the relationship between CSK structure and cell function, but also to identify agents that can be used to regulate functions related to the CSK. Agents commonly used to alter the structure of the CSK are cytotoxic and destructive. Such agents include cytochalasin D (or B) to disrupt actin microfilaments, nocodazole or colchicine to disrupt microtubules and acrylamide to disrupt intermediate filaments.
Control of the integrity of the ECM is believed to be a balance of proteases and anti-proteases. Urokinase (uPA), a plasminogen activator can play a regulatory role in the breakdown of ECM [F. Blasi, BioEssays, 15:105 (1993)]. uPA can cleave at least two precursor proteins: plasminogen to form plasmin, pro-hepatocyte growth factor to form hepatocyte growth factor [K. Dano et al., Adv. Cancer Res., 44:139 (1985); L. Naldini, et al., EMBO, J., 11:4825 (1992)]. Transformed cells and tumor cells have been shown to secrete increased amounts of urokinase plasminogen activator (uPA) [F. Blasi, Bioessays, 15:105 (1993); P. H. A. Quax, et al., Fibrinolysis, 6:41(1992); P. H. A. Quax, et al., Cell Regulation, 2:793 (1991); J. Pollanen, et al., Cancer Res., 57:273 (1991); A. P. Sappino, et al., J. Cell Biol., 109:2471 (1989); J. F. Cajot, et al., J. Cell Biol., 109:915 (1989); V. H. Hearing, et al., Cancer Res., Cancer Res., 48:139 (1988); L. Ossowski, J. Cell Biol., 107:2437 (1988); K. Dano, et al., Adv. Cancer Res., 44:139 (1985); A. P. Sappino, et al., J. Clin. Invest., 88:1074 (1991)]. The role of uPA in cell invasion is thought to involve activation of plasminogen and the subsequent formation of plasmin, as well as degradation of extracellular matrix (ECM). Thus, uPA can help cells to loosen adhesions to ECM, thereby permitting invasion of other tissues. The activity of plasmin and that of uPA are correlated with tumor invasion and metastasis, while inhibitors can block the ECM degradation by tumor cells [P. H. A. Quax et al., Fibrinolysis, 6:41 (1992); A. P. Sappino, et al., J. Clin. Invest. supra; L. Ossowski, et al., Cell, 35:611 (1983); J. F. Cajot, et al., Proc. Natl. Acad. Sci. U.S.A., 87:6939 (1990); L. Ossowski, et al., Cancer Res., 51:274 (1991); B. L. Bergman, et al., Proc. Natl. Acad. Sci. U.S.A., 83:996 (1986)]. Furthermore, in normal cells, the uPA receptor (uPAR) and uPA have also been found at the leading edge of migrating monocytes [A. Estreicher, et al., J. Cell Biol., 111:783 (1990)]. Very recently, cell adhesion in myeloid cells was increased when the uPA/uPAR system was blocked independent of its proteolytic function (D. A. Waltz, et al., J. Clin. Invest., 91:1541 (1993)]. The role of the uPAR-uPA system in cell movement suggests a possible association of the uPA enzymatic system with the cytoskeleton (CSK) [Frixen and Nagamine, Cancer Res., 33:3618-3621 (1993); Jensen and Wheelock, Exp. Cell. Res., 202:190-198 (1992)]. However, no method has been developed which allows regulation of the CSK via the uPA system.
Decontrolled cell growth, decreased cell adhesion, increased cell invasion, and transformed differentiation are a hallmark of malignant transformation. While research data have shown that there are many causative agents [Tomatis, WHO, Internat. Agency for Research on Cancer, Sci. Publ. 100, Lyon, France, (1990)], there are few effective methods which can be used to inhibit abnormal cell growth or to decrease cancer metastasis. Currently, commonly-used methods for inhibiting abnormal cell growth include cell killing with toxins conjugated with monoclonal antibodies to target cancer cells directly or with recombinant toxins, intervening signaling pathways for growth factors, activate or insert tumor suppressor genes, chemotherapy, antiviral therapy [Henderson, et al., Science, 254:1131 (1991); Weinberg, Science, 254:1138 (1991); Aaronson, Science, 254:1146 (1991); Solomon, et al., Science, 254:1153 91991); Adams and Cory, Science, 254:1161 (1991); Hausen, Science, 254:1167 (1991); Pastan and Fitzgerald, Science, 254:1174 (1991)]. Other indirect ways to inhibit abnormal cell growth is the inhibition of anglogenesis to deprive cancer cells of nutrients [Ingber, et al., Nature, 348:555 (1990)]. However, there are very few methods which target specifically intracellular primary structures (the cytoskeleton) to inhibit cell growth and/or cell invasion.
It would be desirable to have a method which can be used to detach adherent cells cultured in vitro without the use of proteases.
It would be desirable to have a simple method of diagnosis of invasive potential of cells.
It would be desirable to have an assay that could be used in vitro to more rapidly determine the invasive potential of cells.
It would be desirable to have an assay that could be used in vivo to determine invasive potential of cells.
It would be desirable to have a method by which one could measure the change in invasive potential of particular tissues.
It would be desirable to have a method which can be used to regulate cellular cytoskeletal stiffness without the use of cytotoxic agents.
It would be desirable to have a method which can be used to readily screen and select compounds that are capable of regulating cellular cytoskeletal stiffness, and thus can be used to regulate functions related to the CSK, for example, cell growth, mitosis, motility, invasion and differentiation.